Data Sheet 1_Typing of Echinococcus multilocularis by Region-Specific Extraction and Next-Generation Sequencing of the mitogenome.pdf

BackgroundInfection by the fox tapeworm Echinococcus multilocularis may lead to a severe zoonosis in humans, alveolar echinococcosis, which may be fatal if left untreated. Typing is important to understand the epidemiology of this parasite, yet there is limited knowledge on the microdiversity of E. multilocularis on the local scale, since the typing resolution of established methods is restricted. MethodsThe mitogenome of E. multilocularis was used as the target regions to modify, apply and validate the Region-Specific Extraction (RSE) method in combination with Next-Generation Sequencing (NGS). Single Nucleotide Polymorphisms (SNPs) were detected in the mitochondrial DNA (mtDNA) and analysed bioinformatically. To validate the success and the accuracy of the RSE protocol, the mitogenomes of some E. multilocularis isolates were also analysed by the Whole-Genome Sequencing (WGS). ResultsWith the chosen combination of methods, the entire mitogenome (~13 kb) of E. multilocularis could be captured and amplified. The read depth (median ≥ 156X) was sufficient to detect existing SNPs. The comparison of mitogenome sequences extracted by RSE with mitogenome sequences obtained by WGS showed that the accuracy of the RSE method was consistently comparable to direct Whole-Genome Sequencing. ConclusionThe results demonstrate that the RSE method in combination with NGS is suitable to analyse the microdiversity of E. multilocularis at the whole mitogenome level. For the capture and sequencing of large (several kb) genomic regions of E. multilocularis and other applications, this method can be very helpful.**背景** 狐源多房棘球绦虫（Echinococcus multilocularis）感染人类后可引发重症人畜共患病（zoonosis）——泡型棘球蚴病（alveolar echinococcosis），若未接受治疗则可能致死。基因分型研究对阐明该寄生虫的流行病学特征至关重要，但现有分型方法的分辨率存在局限，导致当前对多房棘球绦虫的局地微观多样性认知仍较为有限。 **方法** 本研究以多房棘球绦虫的线粒体基因组（mitogenome）为靶标区域，对区域特异性提取法（Region-Specific Extraction, RSE）进行优化、应用与验证，并结合下一代测序（Next-Generation Sequencing, NGS）开展相关实验。研究人员对线粒体DNA（mitochondrial DNA, mtDNA）中的单核苷酸多态性（Single Nucleotide Polymorphisms, SNPs）进行了检测与生物信息学分析。为验证RSE实验流程的有效性与准确性，本研究同时采用全基因组测序（Whole-Genome Sequencing, WGS）对部分多房棘球绦虫分离株的线粒体基因组进行了分析。 **结果** 采用本研究选定的方法组合，可成功捕获并扩增多房棘球绦虫的完整线粒体基因组（约13 kb）。测序深度（中位数≥156倍）足以检测到存在的单核苷酸多态性。将RSE法提取的线粒体基因组序列与WGS获得的序列进行比对后发现，RSE法的准确性始终与直接全基因组测序相当。 **结论** 本研究结果证实，结合下一代测序的RSE法适用于在完整线粒体基因组层面分析多房棘球绦虫的微观多样性。该方法可有效用于多房棘球绦虫大片段（数kb）基因组区域的捕获与测序，亦可推广至其他同类研究应用场景。